本文選題：生育三烯酚 + 人結(jié)腸癌細(xì)胞SW620��； 參考：《天津醫(yī)科大學(xué)》2013年碩士論文

【摘要】：目的 本研究旨在通過體外培養(yǎng)人結(jié)腸癌SW620細(xì)胞、HCT-8細(xì)胞,利用分子生物學(xué)實驗技術(shù),通過多個角度證實生育三烯酚誘導(dǎo)結(jié)腸癌細(xì)胞死亡的死亡方式為paraptosis樣死亡,并以paraptosis樣死亡方式為中心,探討生育三烯酚誘導(dǎo)結(jié)腸癌細(xì)胞發(fā)生的機制。 方法 1.生育三烯酚誘導(dǎo)人結(jié)腸癌細(xì)胞死亡的方式研究 體外培養(yǎng)人結(jié)腸癌細(xì)胞,加入不同劑量的6-生育三烯酚(1、2.5、5、10、15、20、25、30和40μmol/L)和乙醇(溶劑對照)作用24h后,以MTT實驗觀察δ-生育三烯酚對SW620細(xì)胞存活率的影響；按照5、10、15、20μmol/L δ-生育三烯酚作用于細(xì)胞,以熒光染色(AO/EB染色,DAPI染色)觀察細(xì)胞死亡情況。 按照20μmol/L劑量加入δ-生育三烯酚,通過加入paraptosis抑制劑放線菌酮觀察其對生育三烯酚作用的影響情況,來正面證實生育三烯酚誘導(dǎo)人結(jié)腸癌細(xì)胞死亡的方式中存在paraptosis樣死亡。 以不同劑量的δ-生育三烯酚作用于人結(jié)腸癌細(xì)胞SW620中,檢測細(xì)胞中caspase-3的活性,并用抗癌陽性藥物紫杉醇作為腫瘤細(xì)胞凋亡誘導(dǎo)劑作用于SW620細(xì)胞中,利用DNA Ladder方法檢測細(xì)胞中DNA的降解情況,從而證實生育三烯酚誘導(dǎo)人結(jié)腸癌細(xì)胞死亡并非典型凋亡方式。利用免疫細(xì)胞化學(xué)檢測細(xì)胞中細(xì)胞自噬標(biāo)記蛋白LC3的表達情況來觀察生育三烯酚誘導(dǎo)人結(jié)腸癌細(xì)胞產(chǎn)生的這種空泡化死亡現(xiàn)象與細(xì)胞自噬的關(guān)系,從而排除細(xì)胞自噬的可能,從反面證實生育三烯酚誘導(dǎo)人結(jié)腸癌細(xì)胞死亡的方式為paraptosis樣死亡。 為了排除細(xì)胞系的特異性,本實驗中增加了另一種人結(jié)腸癌細(xì)胞系,HCT-8細(xì)胞。將γ-生育三烯酚作用于HCT-8細(xì)胞,觀察細(xì)胞死亡情況,同時檢測細(xì)胞中caspase-3的活性,作為細(xì)胞系平行實驗。 2.δ-生育三烯酚誘導(dǎo)人結(jié)腸癌細(xì)胞SW620死亡的機制研究 體外培養(yǎng)SW620細(xì)胞,加入的20μmol/Lδ-生育三烯酚作用24h后,通過免疫細(xì)胞化學(xué)和western bloting的方法檢測細(xì)胞內(nèi)質(zhì)網(wǎng)膜蛋白標(biāo)志物鈣連蛋白和內(nèi)質(zhì)網(wǎng)應(yīng)激標(biāo)記蛋白Bip的表達情況,檢測6-生育三烯酚誘導(dǎo)人結(jié)腸癌細(xì)胞出現(xiàn)空泡化死亡的現(xiàn)象與內(nèi)質(zhì)網(wǎng)應(yīng)激的關(guān)系；在SW620細(xì)胞中加入wnt通路抑制劑FH535抑制細(xì)胞中wnt通路的激活,觀察細(xì)胞的形態(tài)學(xué)變化,并利用western bloting方法檢測wnt通路關(guān)鍵因子β-catenin的表達量變化,檢測6-生育三烯酚誘導(dǎo)人結(jié)腸癌細(xì)胞死亡與Wnt通路的關(guān)系。 結(jié)果 1.與乙醇對照組相比,6-生育三烯酚作用于結(jié)腸癌細(xì)胞SW62024h后,各劑量組均可以使SW620細(xì)胞存活率降低(p0.05),且細(xì)胞存活率隨著6-生育三烯酚劑量的增加呈現(xiàn)降低的趨勢,IC50值為15.18μmol/L。γ-生育三烯酚作用于結(jié)腸癌細(xì)胞HCT-824h后,各劑量組均可以使HCT-8細(xì)胞存活率降低(p0.05),且細(xì)胞存活率隨著γ-生育三烯酚劑量的增加呈現(xiàn)降低的趨勢,IC50值為32.69mol/L 2.生育三烯酚作用于結(jié)腸癌細(xì)胞SW620和HCT-8后,細(xì)胞內(nèi)均未見細(xì)胞核不規(guī)整,核染色體DNA濃縮、著邊呈新月狀,凋亡小體生成等典型凋亡現(xiàn)象,取而代之的是細(xì)胞內(nèi)出現(xiàn)大量空泡。6-生育三烯酚所致的SW620細(xì)胞死亡可被paraptosis抑制劑放線菌酮所抑制。紫杉醇可使SW620細(xì)胞核內(nèi)DNA產(chǎn)生梯度斷裂,而6-生育三烯酚并不能使SW620細(xì)胞核內(nèi)DNA發(fā)生梯度降解現(xiàn)象。細(xì)胞自噬標(biāo)記物L(fēng)C3表達為陰性。證明6-生育三烯酚誘導(dǎo)結(jié)腸癌細(xì)胞SW620死亡的方式為paraptosis樣死亡而非典型凋亡或細(xì)胞自噬。 3. FH535可導(dǎo)致SW620細(xì)胞死亡,形態(tài)學(xué)顯示細(xì)胞胞漿內(nèi)出現(xiàn)大量空泡,蛋白定量結(jié)果顯示wnt通路關(guān)鍵因子β-catenin的表達量下降。證明生育三烯酚誘導(dǎo)結(jié)腸癌細(xì)胞paraptosis樣死亡與抑制細(xì)胞中Wnt通路的激活有關(guān)。 4.免疫細(xì)胞化學(xué)結(jié)果顯示,δ-生育三烯酚作用于結(jié)腸癌細(xì)胞SW62024h后,內(nèi)質(zhì)網(wǎng)膜鈣連蛋白表達陽性,隨著生育三烯酚劑量的增大,細(xì)胞內(nèi)質(zhì)網(wǎng)逐漸形成空泡。內(nèi)質(zhì)網(wǎng)應(yīng)激標(biāo)記物Bip蛋白表達量增高。證明生育三烯酚誘導(dǎo)結(jié)腸癌細(xì)胞paraptosis樣死亡與細(xì)胞內(nèi)質(zhì)網(wǎng)應(yīng)激有關(guān) 結(jié)論 1.證實生育三烯酚誘導(dǎo)結(jié)腸癌死亡的死亡方式中存在paraptosis樣死亡。 2.發(fā)現(xiàn)6-生育三烯酚對結(jié)腸癌細(xì)胞中wnt信號通路的影響以及引起細(xì)胞內(nèi)質(zhì)網(wǎng)應(yīng)激可能是其誘導(dǎo)SW620細(xì)胞paraptosis樣死亡的機制。
[Abstract]:Purpose

The aim of this study was to investigate the mechanism of tocotrienol - induced apoptosis in colon cancer cells by means of molecular biology experiments by culturing human colon cancer SW620 cells and HCT - 8 cells in vitro .

method

1 . Method for inducing human colon cancer cell death by tocotrienol

The effect of delta - tocotrienol on the survival rate of SW620 cells was observed by MTT assay after 24 h of incubation with 6 - tocotrienol ( 1 , 2.5 , 5 , 10 , 15 , 20 , 25 , 30 and 40 渭mol / L ) and ethanol ( solvent control ) in vitro .
The cell death was observed by fluorescence staining ( AO / EB staining , DAPI staining ) in the presence of 5 , 10 , 15 , 20 渭mol / L 未 - tocotrienol .

未 - tocotrienol ( 未 - tocotrienol ) was added at 20 渭mol / L dose , and the effect of tocotrienol on tocotrienol was observed by the addition of antimicrobial agents . The positive effect of tocotrienol on the death of human colon cancer cells was confirmed .

The activity of caspase - 3 in human colon cancer cells was detected with different doses of delta - tocotrienol , and the activity of caspase - 3 in human colon cancer cells was detected by using anti - cancer positive drug taxol as inducer of tumor cell apoptosis .

In order to exclude the specificity of cell lines , another human colon cancer cell line , HCT - 8 cells was added to the experiment .

2 . Mechanism of 未 - tocotrienol - induced death of human colon cancer cell SW620

SW620 cells were cultured in vitro . After 24 h of 20 渭mol / L 未 - tocotrienol , the expression of calcium connexin and endoplasmic reticulum stress marker protein Bip in human colon cancer cells were detected by immunohistochemistry and western bloting , and the relationship between apoptosis and endoplasmic reticulum stress was detected in 6 - tocotrienol - induced human colon cancer cells .
wnt pathway inhibitor was added to SW620 cells to inhibit the activation of wnt pathway in the cells , to observe the morphological changes of the cells , and to detect the change of the expression of 尾 - catenin in wnt pathway by western bloting method , and the relationship between the death of human colon cancer cells and Wnt pathway was detected by 6 - tocotrienol .

Results

1 . Compared with the ethanol control group , 6 - tocotrienol can decrease the survival rate of SW620 cells ( P < 0.05 ) , and the cell survival rate decreases with the increase of 6 - tocotrienol . The IC50 value is 15.18 渭mol / L . 緯 - tocotrienol acts on colon cancer cells HCT - 824h , and the cell survival rate decreases with increasing the dose of 緯 - tocotrienol , the IC50 value is 32.69mol / L .

2 . When tocotrienol acts on colon cancer cells SW620 and HCT - 8 , there is no abnormal cell nucleus in the cells . The DNA concentration of nuclear chromosomes , the formation of apoptotic bodies , and the like are inhibited . The death of SW620 cells induced by 6 - tocotrienol can be inhibited .

3 . The cell death of SW620 cells could result in the death of SW620 cells , and a large amount of vacuoles appeared in the cytoplasm of the cells . The results showed that the expression of 尾 - catenin was decreased in the wnt pathway .

4 . Immunocytochemistry showed that 未 - tocotrienol acts on colon cancer cells SW620 h after SW620 h . The expression of 尾 - tocotrienol increased with the increase of tocotrienol dose . The expression of 尾 - tocotrienol increased with the increase of tocotrienol dose . The expression of Bip protein in the endoplasmic reticulum stress marker was increased . It was shown that the death of the tocotrienol - induced colon cancer cells was related to the stress of the endoplasmic reticulum stress .

Conclusion

1 . It was confirmed that the death of a tocotrienol - induced death of colon cancer was fatal .

2 . It was found that the effects of 6 - tocotrienol on the wnt signal pathway in colon cancer cells and the possible mechanism of inducing the death of SW620 cells in SW620 cells .

【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R114;R735.35

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本文編號：1761525